The surface change was plotted on a logarithmic scale and normalized to the culture time to obtain the doubling per day. To model the cumulative cell number of nonmutant (NM) and mutant (M) EP populations, their cell population doubling rate per passage (D), noted as Dand Dis the mean D of the first five EPs (the adaptation phase) (EP1 to EP5, starting from P13 for HD291 and from P25 for HS306 cells), whereas Dis the mean D of five EPs during the plateau phase of growth (P13+EP30 to P13+EP34 for HD291 and P25+EP10 to P25+EP14 for HS306 cells). before switching to enzymatic single-cell passaging. We show that karyotype abnormalities and copy number variations are not restricted to long-term culture, but can occur very rapidly, within five passages after switching hESCs to enzymatic dissociation. Subchromosomal abnormalities preceded or accompanied karyotype abnormalities and were associated BAY 293 with increased occurrence of DNA double-strand breaks. Our results indicate that enzymatic single-cell passaging can be highly deleterious to the hPSC genome, even when used only for a limited period of time. Moreover, hPSC culture techniques should be reappraised by complementing the routine karyotype analysis with more sensitive techniques, such as microarrays, to detect subchromosomal abnormalities. Introduction Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are BAY 293 pluripotent stem cells (PSCs) that can be indefinitely maintained in culture in adequate conditions. At any time point, PSCs can be induced to differentiate for research or clinical objectives. Moreover, they proliferate constantly in vitro, providing means for massive cell expansion, a unique feature for normal cells. However, the long-term genomic integrity of PSCs in culture is a major concern [1C4]. Indeed, hESCs generally display a normal karyotype at derivation; however, karyotypic abnormalities, particularly gain of chromosome 1, 12, 17, and 20, may arise after prolonged in vitro culture. Subchromosomal changes were also reported suggesting that PSC genomic instability BAY 293 may be underestimated [5]. These genomic abnormalities are a serious concern not only for PSC medical applications, but also for their use in biomedical research [1,6]. PSC genomic instability during in vitro culture has raised numerous concerns about the effects of culture conditions. Indeed, the currently used human PSC (hPSC) culture methods are based on various culture media, culture feeders/matrices, or passaging techniques that may differently affect genetic stability or impart different selective pressure on cells. However, the effect BAY 293 of these variables on the incidence of PSC genomic defects has never been systematically assessed. The question of the optimal passaging technique is representative of this issue. Several reports suggest that enzymatic passaging (EP) [3,5,7C9] or feeder-free culture conditions [5, 10] might lead to more frequent cytogenetic aberrations, but the impact of culture conditions on the genetic integrity of hPSCs remains debated [11,12]. Rabbit Polyclonal to OR10H2 Conversely, other works show that mechanical passaging might favor the appearance of cytogenetic abnormalities, such as amplification of the 20q11.21 segment [4,13]. Indeed, mechanical passaging may lead to a selection bias based on the colony morphology and some recurrent aneuploidies could result in the accentuation of pluripotency-associated features, such as reduction of spontaneous differentiation and more robust colony expansion [14]. While the resolution of the genomic abnormality detection methods and the number of studied hPSC lines increase, several crucial questions remain still unanswered, such as the rate at which such abnormalities accumulate and the contribution of the different culture conditions. Therefore, we precisely monitored genomic abnormalities in hESC lines, cultured in parallel, using mechanical and single-cell EP techniques up to 45 passages. Our results demonstrate that the use BAY 293 of single-cell passaging can be deleterious to PSC genome integrity and that subchromosomal and karyotype abnormalities can be detected just after five passages. Materials and Methods Cell culture The human HD129, HD291, and HS306 ESC lines were derived as previously reported [15,16]. Cells were maintained in standard KO culture medium: 80% KO-DMEM, 20% KOSR, 2?mM L-glutamine, 1% nonessential amino acids, 0.5?mM -mercaptoethanol (all from Gibco Invitrogen), supplemented with 10?ng/mL bFGF (Abcys). Cells were grown in 35-mm dishes that were precoated with irradiated (40 Gy) human foreskin fibroblast feeders (40,000 cells/cm2) and were either enzymatically or mechanically passaged every week. Mechanical passaging (MP) was carried out under an inverted microscope in a hood using scalpels [17,18]. Undifferentiated cell clumps were excised and plated on freshly prepared (as above) feeders. For EP, cells were pretreated with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 for 1?h and dissociated with TrypLE? Select (Invitrogen) at 37C for 10?min. The reaction was stopped.